An increasing number of specific antigens from different types of organisms (e.g. tumor cells, bacteria, virus and parasites) has been produced using cloning techniques over the last years. However, these antigens are frequently weak immunogens despite their high specificity.
To obtain good protection after vaccination, immune stimulating systems are needed that can enhance and activate the immune system against these weak antigens. Such immune stimulating systems are called adjuvants.
Adjuvants, presently mainly used in animal experiments, includes a highly heterogeneous group of substances; inorganic substances, oil emulsions, charged polymers, neutral substances or substances from bacteria.
There are presently large efforts in research and development in order to obtain a safe adjuvant with high efficacy to be used in humans. However, today there is presently no general adjuvant for this purpose.
Alum hydroxides and alum phosphates were the first two inorganic substances that were used in humans. The immune response obtained is a result of slow desorption of the precipitated antigen on the surface of the particle. Later it was shown that phagocyting cells were attracted by these alum salts leading to further enhancement of the immune response. However, these salts are not safe since granuloma formation has been reported (Slater et al, Br. J. Dermatol. (1982) Vol. 107, page. 103–108.). Furthermore, the alum salts can not be used for all antigens since all antigens are not adsorb on the surface.
In 1944 Freund introduced his adjuvant consisting of a mixture of vegetable oil, mineral oil, detergents and killed bacteria. The enhancement obtained was partly due to slow release of the antigen from the oil emulsion. Freunds adjuvant can however not be used in humans due to granuloma formation, induction of auto-immune reactions and the non-biodegradable mineral oil. Furthermore, the effect is difficult to control. The active substance in Freunds adjuvant has been isolated and its structure determined and shown to be N-acetyl muramyl-L-alaninisoglutamate, often called muramyl-dipeptide (MDP).
The adjuvant effect dependent of the particle size of polymetacrylate and polystyrene particles was examined on mice (Kreuter et al, Vaccine, (1986) vol 4, 125–129) by the use of ovalbumin (adsorbed on the particles) as a model antigen with subsequent assay of the immune response. The size of the particles was varied between 62 and 306 nm. The result was that smaller particles enhanced the immune response better than larger. The smaller particles gave a better effect than 0.2% Al(OH)3. All preparations elicited a higher response as compared to fluid preparations. Similar experiments where particulate systems with smaller size results in a higher immune response as compared to larger particles are known in the scientific literature.
Almost all systems used today for enhancement of the immune response against antigens are particles or is forming particles together with the antigen. In the book “Vaccine Design—the subunit and adjuvant approach” (Ed: Powell & Newman, Plenum Press, 1995) all known adjuvants are described both regarding their immunological activity as well as regarding their chemical characteristics. As described in the book more than 80% of the adjuvants tested today are particles or polymers that together with the antigens (in most cases proteins) are forming particles. The type of adjuvants that not are forming particles are a group of substances that are acting as immunological signal substances and which under normal conditions consists of the substances that are formed by the immune system as a consequence of the immunological activation after administration of particulate adjuvant systems.
Using particulate systems as adjuvants, the antigens are associated or mixed with or to a matrix which has the characteristics of being slowly biodegradable. Of great importance using such matrix systems are that the matrix does not form toxic metabolites. Choosing from this point of view, the main kind of matrices that can be used are mainly substances originating from a body. With this background there are only a few systems available that fulfils these demands: lactic acid polymers, poly-amino acids (proteins), carbohydrates, lipids and biocompatible polymers with low toxicity. Combinations of these groups of substances originating from a body or combinations of substances originating from a body and biocompatible polymers can also be used. Lipids are the preferred substances since they display structures that make them biodegradable as well as the fact that they are the most important part in all biological membranes.
Lipids are characterized as polar or non-polar. The lipids that are of most importance in the present invention are the polar lipids since they have the capacity to form particulate systems in water. Another way of defining these lipids are as amphifilic due to their chemical structure with one hydrophobic and one hydrophilic part in the molecule thereby being useable as surface active substances. Examples of main groups of polar lipids are mono-glycerides, fatty acids, phospholipids and glycosphingolipids. These main groups can be further characterized depending on the length of the acyl chain and the degree of saturation of the acyl chain. Since the number of carbon atoms in the acyl chain can be in the range of 6 to 24 and the number of unsaturated bonds can be varied there are an almost infinite number of combinations regarding the chemical composition of the lipid.
Particulate lipid systems can be further divided into the different groups as discussed in the scientific literature such as liposomes, emulsions, cubosomes, cochleates, micelles and the like.
In a number of systems the lipids may spontaneously form, or can be forced to form, stabile systems. However, under certain circumstances other surface active substances has to be introduced in order to achieve stability. Such surface active systems can be of non- lipid character but possess the characteristics of the polar lipids having hydrophobic and hydrophilic parts in their molecular structure.
Another factor that has been shown to be of importance is that lipids exhibit different physical chemical phases, these phases has in different test systems been shown to enhance uptake of biological substances after administration to mucosal membranes.
In the classical immunology and in combination with vaccination against different types of infectious agents e.g. bacteria, virus or parasites the prevailing dogma has been to administrate the vaccine subcutanously or intramuscularly. However, research has during the last years shown that the body has a very effective immunological system that resides in the mucosa. It has been shown that you can administrate vaccines orally, nasally, rectally and vaginally. In the same way as for the classical immunization it has been shown that by mucosal vaccination there is also a need for enhancement of the immunological response by the addition of adjuvants.
In the same way as within the classical immunology where vaccines (antigens) are administrated parenterally, there is within mucosal immunization a great interest in directing the immunological response towards development of humoral and/or cellular response. If you obtain a humoral response it would be important to direct the response in a way that a certain class of antibodies would be obtained. In order to obtain such a goal, specific immune stimulating agents can be added to the formulation of antigens and adjuvants.
Different types of immune stimulating substances are available. One type is represented by proteins e.g. PHA, Con A, SEA or different types of interferons or interleukines. Another type of substance is represented by MDP, as mentioned above. Additional groups can be characterized as lipid derivatives since they show molecular structures which are amphiphilic. One example of such a substance is called MPL. Another similar substance is Quil A. A number of substances that can be classified within these categories are described in the book “Vaccine Design—the subunit and adjuvant approach” as discussed above.
It would be extremely valuable to be able to make the immunization procedures more effective directing the immunological response towards a certain class or subclass of antibodies and/or to be able to induce a strong T-cell response against the antigens.